Why is the large molecular weight in the back during electrophoresis, while the column is in the fro

Electrophoresis is performed according to the molecular weight of the separated substance and the difference in charge. During electrophoresis, the large molecular weight moves slowly, and the small molecular weight moves fast, so after electrophoresis for a period of time, the large molecular weight is behind.

For chromatographic columns, the owner should refer to gel filtration (there are many types of chromatographic columns, such as ion exchange chromatography, affinity chromatography, etc., the principle of separation is different), which is based on the size of the separated substance. To put it simply, the substances filled in the gel filtration contain many small pores, and among the substances to be separated, the large molecular weight does not enter the small pores, and flows out directly with the eluent, and the small substances enter these small pores, shuttle through these air, naturally, the eluent flows out after elution.

The molecular weight is at the back during electrophoresis because of its large molecular weight, and the amount of electricity provided is equal mv=mv for each molecule

m>m, so vThe < V molecular weight is larger in the back. The column has a large molecular weight in the front, because the molecular weight is large and the volume is large, it is difficult to pass through the frit plate (filter), and the small one can, so it is on top (in the front) < p>

Agarose. It is a linear polysaccharide purified from agar and linked by D-galactose and 3,6 anhydrated L-galactose. The agarose gel is made by placing a dry agarose suspension buffer.

, the usual concentration is 1%-3%, heated and boiled until the solution becomes clear, injected into the template and then cooled at room temperature to condense that is, the agarose gel. Agarose is used as a solid support matrix in DNA backup electrophoresis. The agarose has a relatively stable cross-linked structure in intramolecular and intermolecular hydrogen forms, which makes the agarose gel have better anti-convection properties.

The wells of the agarose gel can be controlled by the initial concentration of the agarose, with lower concentrations of agarose-shaped wells and smaller agarose-shaped wells. Although agarose itself has no electric charge, some glycans may be carboxylated.

Methoxy groups are especially sulfates.

Different degrees of substitution make the surface of the agarose gel have a certain charge, which causes electroosmosis and electrostatic interaction between the sample and the gel during electrophoresis, which affects the separation effect.

Agarose gels can be used as electrophoresis support media for proteins and nucleic acids, and are especially suitable for the purification and analysis of nucleic acids. For example, the pores of the agarose gel with a concentration of 1% are relatively large for the protein, and the hindrance effect on the protein is small, so the influence of the protein molecular size on the electrophoretic mobility is relatively small, so some electrophoresis techniques that ignore the size of the protein and only separate it according to the natural charge of the protein, such as immunoelectrophoresis, electrofocusing electrophoresis such as plates, etc. Agarose is also suitable for the separation and analysis of DNA and RNA molecules, because DNA and RNA molecules are usually large, so there will be a certain friction hindrance in the separation, and the size of the molecule will have a significant impact on the electrophoretic mobility.

For example, for double-stranded DNA, the size of the electrophoretic mobility is related to the size of the DNA molecule, but to the base.

Arrangement and group none. In addition, some agarose with a low melting point melts at 62 °C, and the sample (e.g., DNA) can be re-dissolved into solution by leaving it in a water bath heated to the melting point for a period of time.

Because the elasticity of agarose gels is poor, it is difficult to remove from the tube, so the general agarose gel is not suitable for tubular electrophoresis, and tubular electrophoresis usually uses polyacrylamide gels. Agarose gels are usually aqueous plate gels that are electrophoresis for proteins such as isoelectric focusing and immunoelectrophoresis, as well as for the analysis of DNA and RNA. Vertical electrophoresis is used relatively rarely.

Columns. It's troublesome, it's hard to explain, see for yourself:

How is the vapor phase method made???

A centralized post for gas phase data.

Three-valve (six-way valve) four-column injection separation principle (tandem).

Gas chromatography common sense.

Principles of gas chromatography separation.

Principles of Meteorological Chromatography:

Schematic demonstration of the chromatographic process.

Principles of chromatography (there are many in this post that should have what you need).

Gas Chromatography Basics – Fundamentals (Free PPT).

Sincerely hope to be able to help you.

The inertia of a large mass is large, and the inertia of a small mass is small.

Of course, it is the first peak with a large expansion volume, and the branching expansion volume is large.

The glue resolution is not enough When the large fragment is moving, the speed is very slow, the distance between the fragments cannot be stretched, and the band cannot be seen clearly by the naked eye.

For very large molecular weight fragments, pulsed field gel electrophoresis can be used. It is also possible to extend the electrophoresis time (at which point the small DNA in front may run out of the gel. If you're only looking for large fragments, it's still possible to extend the electrophoresis time.

After all, pulsed field gel electrophoresis requires special equipment and is troublesome.

Generally, large molecular weights will have some tailing, but it will not be so severe. There may be some reasons for this: 1. The electrophoresis voltage is too high; 2. The buffer is left for too long; 3. The gel quality is not ideal (this is not very likely for your result); 4. The gel concentration is high.

It should be that the large fragment is easy to degrade, and you have been running for a long time, so the buffer generally needs to be new and pay attention to the contamination of DNase, and there is not a concentration of the sample is too high, you can try the gradient of geometric multiple dilution. Of course, if you want to run macromolecules, it is necessary to reduce the concentration of glue.

Gel columns and SDS are inherently different.

As a sample solution containing various molecules flows slowly through a gel column, each molecule moves simultaneously in two different motions within the column: a vertical downward movement and an undirected diffusion movement. Due to its large diameter, macromolecular substances are not easy to enter the micropores of gel particles, but can only be distributed between particles, so they move downward quickly during elution.

In addition to diffusion in the gap between gel particles, small molecule substances can also enter the micropores of gel particles, that is, into the gel phase, in the process of moving downward, from one gel to the particle gap and then into another gel particle, so that the downward speed of small molecule substances lags behind that of large molecules, so that the large molecules in the sample flow out of the column first, the medium molecules flow out, and the smallest molecules flow out last. As for how small the molecules can be hindered by the gel particles, it is related to the pore size of the molecular sieve.

SDS-PAGE generally uses a discontinuous buffer system, which can have a higher resolution than a continuous buffer system. The function of the stacking gel is to have a packing effect, the gel concentration is small, the pore size is large, the thinner sample is added to the stacking gel, and the migration of the large pore size gel is concentrated into a narrow zone. When the sample solution and stacking gel are selected for Tris HCl buffer, the electrode solution is selected for Tris glycine.

After electrophoresis begins, HCl dissociates into chloride ions, and glycine dissociates a small amount of glycine root ions. The proteins are negatively charged, so they move towards the positive electrode together, where the chloride ion is the fastest, the glycine ion is the slowest, and the protein is centered. At the beginning of electrophoresis, the chloride ion swim rate is the largest, exceeding that of the protein, so a low conductance region is formed in the back, and the electric field strength is inversely proportional to the low conductance region, thus producing a higher electric field strength, so that the protein and glycine ions move rapidly, forming a stable interface, so that the protein gathers near the moving interface and concentrates into an intermediate layer.

In this identification method, the mobility of a protein is determined primarily by its relative molecular mass, independent of its charge and molecular shape.

I think it has something to do with the pore size of the zeolite, the sds-page has a larger pore size, so no matter what the molecular weight is, it goes through the pore, so in the end, the big protein is left behind, and the gel chromatography has a smaller pore size, and the mass protein doesn't go through the pore size, but through the gap in the pore, and the path is much shorter, so the big one comes out first

I don't know if that's the case

I don't know exactly what you're measuring a substance, but if you have the same molecular weight, you can't tell the difference.

Agarose gels can be used to destroy the support medium for protein and nucleic acid electrophoresis, which is especially suitable for the purification and analysis of nucleic acids. For example, the pores of the agarose gel with a concentration of 1% are relatively large for the protein, and the blocking effect on the protein is small, so the influence of the protein molecular size on the electrophoretic mobility is relatively small, so some electrophoresis techniques that ignore the size of the split-wheel protein and only separate the protein according to the natural charge of the protein are applicable

Oh, generally small molecules are measured at about 100A, and 300A for ordinary macromolecules, and the general pore size should be slightly greater than or equal to 3 times the molecular diameter.

Is there a picture? What brand? Ask the supplier's technical support directly. It could be a product issue.

What electrophoresis do you do? What kind of glue is used? It may be that the ratio of glue is not enough to separate large molecular weights.

Nowadays, most protein markers can be directly electrophoresed and do not need to be reduced.

Is there a reduce (I don't know how to translate it)? If there is no reduce, the polar amino acid residues on the outer surface of the native structure of the protein molecule will not be enough to provide the necessary amount of charge for electrophoresis.